Comparative biological activity of abamectin formulations on root-knot nematodes (Meloidogyne spp.) infecting cucumber plants: in vivo and in vitro

The root-knot nematodes (Meloidogyne spp.) are considered one of the most destructive diseases in the world. In Egypt, farmers primarily rely on chemical nematicides, which have become costly to control. Currently, abamectin is a bio-based pesticide used as an alternative tool against Meloidogyne spp. on cucumber plants (Cucumis sativus L.). During the current research, four tested abamectin formulations were DIVA (1.8% EW), RIOMECTIN (5% ME), AGRIMEC GOLD (8.4% SC) and ZORO (3.6% EC) compared with two reference nematicides namely, CROP NEMA (5% CS) and TERVIGO (2% SC). The main results showed that, in vitro study elucidated that the most effective formulations of abamectin as a larvicidal were EW with LC50 value of 21.66 µg ml−1. However, in the egg hatching test, the formulations of abamectin SC (2%) and EW were the most effective in reducing egg hatching, with LC50 values of 12.83 and 13.57 µg ml−1. The calculated relative potency values showed diversity depending on the two referenced nematicides. On the other hand, in vivo study, the results indicated that, all tested formulations of abamectin recorded general mean reductions in root galls (23.05–75.23%), egg masses (14.46–65.63%). Moreover, the total population density declined by 39.24–87.08%. Furthermore, the influence of abamectin formulations, in the presence of root-knot nematodes, on the growth of cucumber plants parameters, such as root dry weight, root length, root radius, root surface area, shoot dry weight and shoot height, as well as the content of macro-elements (N, P and K) exhibited varying levels of response.

The tested abamectin formulations and their rates. Six different commercial formulations of abamectin products were selected from the Egyptian markets to be investigated under in vitro and in vivo conditions. The abamectin formulations were DIVA (1.8% EW), RIOMECTIN (5% ME), AGRIMEC GOLD (8.4% SC), ZORO (3.6% EC), CROP NEMA (5% CS) and TERVIGO (2% SC). Noteworthy, there were two SC formulations; the 1st was AGRIMEC GOLD (8.4% SC) and the 2nd was the referenced nematicide TERVIGO (2% SC). Each product was evaluated in two rates: 50 and 100 g a.i./feddan; these rates were established according to the standards registered in Egypt.
In vitro assays. The impact of different abamectin formulations on egg hatching and larval mortality of the root-knot nematodes (Meloidogyne spp.) was assessed under laboratory conditions (29 ± 2 °C). Moreover, several experiments were conducted to establish the effective concentration ranges of abamectin products.
Hatching assays. The tested concentrations of abamectin products were ranged from 2.5 up to 800 µg/ml. The vials (each one ca. 15 ml) containing distilled water served as untreated checks. Each concentration was replicated four times and each replicate included approximately 1000 eggs. The numbers of hatched eggs were recorded and their EC 50 were calculated after 7 days of application.
Mortality assays. The tested concentrations of each abamectin product during this study ranged from 2.5 up to 800 µg/ml. Each concentration was replicated four times, and each replicate included approximately 200 J 2 s. Vials included distilled water served as controls. The numbers of both dead and alive J 2 were recorded after 48 h of exposure, and the mortality percentages were calculated.
In vivo assays (experimental design of microplots). The performance of abamectin products was investigated on cucumber plants infested with root-knot nematodes. The pots were filled with 2 kg of autoclaved sandy soil (pH 8.3, O.M. 0.18%). All abamectin formulations were applied as soil drench at two rates of 50 and www.nature.com/scientificreports/ 100 g a.i./feddan in comparison to two standard products, namely, CROP NEMA (5% CS) and TERVIGO (2% SC). A cucumber plantlet (cv. Dahab) was transplanted into each pot, and 3 days later the inoculation process with 7000 eggs/pot was executed. Two types of untreated controls were used: inoculated (Control +) and uninoculated (Control −). All treatments were replicated five times, and pots were in outdoor conditions (28 ± 2 °C, 70 ± 2 RH and 14: 10 lights: dark period). During the experiment, irrigation and fertilization were applied when appropriate. Sixty-two days later, after the inoculation, the plants were removed and washed to be free of soil. Shoot height, shoot dry weight and root dry weight, in addition to the root length, root radius and root surface area 27 were estimated. The second stage of juveniles (J 2 s) were extracted, and the roots were stained with Phloxine B to facilitate egg mass counting. The number of galls/2 g roots and egg-masses/2 g roots were counted. The total population density was estimated by quantifying and summing individuals of eggs/root system together with the second stage of juveniles/2 kg soil 28 . The macro-elements such as nitrogen (N), phosphorus (P), and potassium (K) were determined in the cucumber tissues at the termination of the experiment in the lab at the department of reclamation and cultivation of desert lands, faculty of Agriculture (Saba-Basha), Alexandria University. However, the cucumber used in this study (cv. Dahab) is formally registered in the Egyptian Ministry of Agriculture.
Statistical analysis and experimental design. The hatching and J 2 s mortality percentages were estimated using the Abbott formula 29 , and Probit analysis was used to calculate the LC 50 for larvae and EC 50 for eggs each compound according to 30 . The relative potency of tested products was calculated according to 31 using the Polo plus program 32 for two references (SC and CS formulations). The statistical analysis of data was carried out using the computer program 33 . The microplots experiment was arranged in a complete randomized (CRD) design with five replications for each treatment, each replicate consisted of one plant. Statistically, the significant differences between the means were compared using analysis of variance (ANOVA) with the least significant differences (LSD) and P values = 0.05 probability.
Ethics approval and consent to participate. This article does not contain any studies with human or animal subjects. The current experimental research including the collection of plant material, is complying with relevant institutional, national, and international guidelines and legislation and used for research and development.
Permission statement. To collect the plant material for this study a permission was obtained from Prof.
El-sayed H. Eshra, the head of plant protection research station, Agricultural Research Center, Alexandria, Egypt. Also, The Agricultural Research Center who's the responsibility for formal identification of the plant material used in our study.

Microplot experiment (in vivo study).
The tested products of abamectin were evaluated at two rates 50 and 100g a.i./feddan. Also, both referenced nematicides; CROP NEMA (5% CS) and TERVIGO (2% SC) were used in comparison at both rates (Figs. 3A-C, 4A-C). The application of abamectin as SC (8.4%) recorded the highest general mean reduction (GMR%) in the total population density (J 2 + eggs) of Meloidogyne spp. followed by SC (2%), CS, ME, EW, and EC with 87.08, 82.87, 76.11, 58.05, 43.82 and 39.24%, successively. The high rate of the formulations (100 g a.i./feddan) recorded the highest reduction percentages compared to the lowest rate (50 g a.i./feddan), except with ME and CS formulations. There are significant differences between the two tested rates except for SC (8.4%) and SC (2%) (Figs. 3A-C, 4A-C).  37.75 and 23.05%, respectively. However, no significant difference was noticed between the high and low rates of EC formulation, while the remaining formulations showed significant differences (Figs. 3A-C, 4A-C).
The egg masses were decreased with all applied treatments (Figs. 3A-C, 4A-C). The application of abamectin as SC (8.4%), CS, EC, SC (2%), ME and EW formulations recorded GMR of 65.63, 58.01, 38.68, 36.53, 32.42 and 14.46%, successively. The significance analysis exhibited that no significant differences were observed between the high and low rates of EC, and SC (2%) formulations.
The effect of abamectin formulations on cucumber growth. The influences of applied abamectin at different formulations on the growth parameters of cucumber plants were recorded (Figs. 5A-C, 6A-C, 7A-C, 8A-C). The recorded plant growth parameters were root dry weight, root length, root radius, root surface area, shoot dry weight and shoot height. In the untreated (uninoculated) plants, the root dry weight was decreased by 8.45%; also, the shoot dry weight and height were decreased by 9.60 and 7.99%, respectively (Figs. 5, 6). The obtained results showed that formulations of ME, CS and EC were the only treatments that recorded general mean increases in root dry weight of 27.22, 14.76 and 13.32%, respectively. While abamectin at SC (8.4%), EW and SC (2%) were decreased the root dry weight by 16.44, 13.40 and 9.68%, respectively. No significant differences were noticed between the high and low rates of EW, ME, SC (8.4%) and CS formulations. Meanwhile, all the formulations of abamectin such as SC (8.4%), ME, CS, EW, and EC, increased the shoot dry weight by 158.71, 26.20, 19.56, 16.65 and 9.69%, respectively, while SC (2%) reduced it by 7.67%. Unfortunately, there are no significant differences were observed between EW, EC, ME, CS and SC (2%) formulations at either high or low rates. Vice versa, all applied formulations of abamectin were minimized the shoot height of cucumber plants by 26.35, 17.17, 9.52, 9.24, 5.05 and 0.17% with CS, SC (8.4%), EC, ME, EW, and SC (2%), respectively. Application of abamectin at EC, CS and SC (2%) showed no significant differences between the high and low rates, while there are significant differences between EW, ME, and SC (8.4%) formulations (Figs. 5, 6).
The efficacy of abamectin formulations in the presence of root-knot nematodes were evaluated on the root length, root radius and root surface area (Figs. 7A-C, 8A-C). The application of abamectin in EC and ME formulations increased the root length by 11.19 and 7.24%, successively. Otherwise, SC (8.4%), SC (2%), CS and EW formulations decreased the root length by 49.77, 47.25, 18.91 and 14.23%, successively. In the same context, the root surface area was increased with EC and ME formulations by 18.37 and 10.45%, consecutively. While  (1) values (Fiducial Limits), and (C) Relative potency (2) values (Fiducial Limits). * Asterisks (*) means significant differences while ( ns ) means not significant. * ( 1 ) and ( 2 )  .00 and 7.02%, consecutively. The abamectin formulation of EC exhibited no significant differences between the higher and lower rates for both root length and root surface area. The root radius of cucumber plants was increased by 13.75, 10.84, 9.59, 7.92, 6.25 and 0.84% with SC (8.4%), EC, SC (2%), CS, EW, and ME formulations, consecutively. Moreover, all the tested formulations of abamectin exhibited no significant differences between the higher and lower rates.

The impact of abamectin formulations on macro elements in cucumber roots. The effects of
infection with the root-knot nematodes and different formulations of abamectin on the content of nitrogen (N), phosphorus (P) and potassium (K) elements in cucumber roots were measured as found in (Fig. 9). The obtained results indicated that the untreated (uninoculated) pots had increases of N and P by 78.57 and 20.00%, respectively.

Disscusion
During the current discussion, we present the main effect of root-knot nematode on cucumber plants and the use of the abamectin formulations as an alternative tool to control the Meloidogyne spp. Root-knot nematode is widely distributed in greenhouses of cucumber production in Egypt. The idealistic root galling symptoms were noticed in either the absence of nematicides or as a result of control failures as reviewed by 34 . Currently, in Egypt, there are few of available options for managing Meloidogyne sp. and the non-chemical control agents are commercially difficult to be available and often unsatisfactory 35 . During this study, various concentrations of abamectin at different formulations, namely, SC, EW, ME, EC and CS, exhibited different levels of mortality  (1) values (Fiducial Limits) and (C) Relative Potency (2) values (Fiducial Limits). * Asterisks ( * ) means significant differences while ( ns ) means not significant. * ( 1 ) and ( 2 )  www.nature.com/scientificreports/ which could be due not only to the active ingredient concentration but also to the adjuvants in the examined products, and these findings agree with 36 .
In accordance with other studies, it was stated that the formulations of abamectin are the key factor in the biological activity against plant parasitic nematodes 23 . Other study by 37 reported that abamectin in the SC formulation is more effective than EW under laboratory conditions. Also, 38 clarified that abamectin as SC at certain doses decreased the soil population of M. incognita infested tomato plants at a range of 23.40-43.29%, while abamectin as EW recorded a reduction at a range of 25.67-34.37%. However, no phytotoxicity was detected for both formulations.
A remarkable reduction in soil population and root gall index of M. incognita was achieved with abamectin (2.5% EC) and cadusafos under pot or field conditions 39 . In pot experiments, abamectin (VERTIMEC 1.8% EC) at 100 and 200 µg/ml against M. incognita on cabbage plants cv. Balady were minimized the galls, which ranged from 40 to 88%, while egg masses ranged from 58 to 98% and these are data in the same line as our results 40 .
In the same context, the superiority of abamectin as SC against the final populations in compared with the remaining formulations may be attributed to the moderate adsorption ratio on soil particles, whereas, the EC formulation had the highest adsorption ratio, which dramatically decreased their mobility in soil 23 . The use of formulations as water-based suspension concentrate (SC) provided environmental, economic, and social advantages, which included the safety to the applicators and the environment, ease of handling, relatively low cost, a high concentration of insoluble active ingredients, and the ability to be built in water-soluble adjuvants for enhanced biological activity 41 .
Meanwhile, Radwan 42 elucidated that abamectin (SC) or emamectin benzoate (WDG) showed high toxicity against the J 2 of M. incognita in vitro. Emamectin and abamectin were succeeded in decreasing galls, egg masses, eggs, and soil population density significantly. The use of abamectin (2% SC) alone is more effective than the It's worth mentioning that our obtained results indicate that some applied formulations of abamectin showed a negative effect on cucumber growth and the content of phosphorus and potassium elements, but many researchers find the opposite. On the other hand, the cucumber yield and plant height had increased significantly with abamectin (5% EC) at low or high rates compared with a crop produced without using abamectin. However, using dazomet or chloropicrin in combination with abamectin exhibited no significant differences in the total crop yield 43 . The tomato fresh weight and height showed the same significance with untreated check when applied abamectin SC or EW 38 . The application of abamectin (2% SC) achieved increases in the shoot and root dry weights of tomato plants by 16.92 and 14.26%, respectively 42 . The total marketable yield of tomatoes and the plant growth were improved when applied abamectin (2.5% EC) and cadusafos were applied against M. incognita at 5, 7.5 and 10 L/ha 39 . Also, the growth of olive plants e.g., the fresh weights and the length of both shoot and root, were increased at a range of 15.5 up to 105.8% over control with application of abamectin against M. incognita in compared with three tested bio-agents under greenhouse conditions 44 .
The pesticides that used during crop production processes inevitably remain in the soil, affecting rhizosphere microorganisms and plant growth as reviewed by 45 . However, during the current study, some treatments of abamectin formulations exhibited decreasing in the growth parameters for cucumber plants and this may be attributed to the residue of abamectin and/ or its metabolites which adversely affect the soil invertebrates and the roots of cucumber, and this finding is in the same line with those obtained by 43,46 . Furthermore, abamectin is degraded to 8a-hydroxyavermectin B1 a which is a low toxic product that may be taken up as a carbon source for microorganisms and then struggle with plant roots on nutrients 20,47 .
In conclusion, abamectin is an effective nematicide that has been recorded to control a wide range of plant parasitic nematodes, for instance, Meloidogyne spp., Rotylenchulus reniformis and Tylenchulus semipenetrans, on different crops in line with the global trend in integrating nematode management 48 . In current study, abamectin has shown a good efficacy in controlling the root-knot nematodes (Meloidogyne spp.). Also, the formulation as SC (8.4%) recorded the highest reductions in total population density (J 2 + eggs), gall formation, and egg masses. However, the EW formulation was the least effective treatment. Current results recommend more research on abamectin formulations to determine their exact biological activity against plant parasitic nematodes under different environmental impacts and to achieve the prospective aims of sustainable agriculture.